Capsular polysaccharides (cps) are important immunogens found on the surface of bacteria involved in various bacterial diseases. This feature has led to them being an important component in the design of vaccines. They have proved useful in eliciting immune responses especially when linked to carrier proteins (Ref. 1).
Typically, capsular polysaccharides are produced using batch culture in complex medium (Group B Streptococcus, Staphylococcus aureus, Streptococcus pneumoniae and Haemophilus influenzae), fed batch culture (H. influenzae) or continuous culture (Group B Streptococcus and Lactobacillus rhamnosus) (Refs. 2-7). Most studies used batch culture systems in which the growth rate, nutrient levels and metabolic concentrations change during incubation. In such systems, alteration of one factor results in changes in other factors associated with growth that can affect yields unpredictably. Continuous cultures allow the researcher to separate and define parameters that are interdependent during batch culture growth, such as growth rate, nutrient and product concentrations and cell density. During continuous culture, fresh medium is added to a culture at a fixed rate and cells and medium are removed at a rate that maintains a constant culture volume. Continuous culture was preferred for capsular polysaccharide production when it proved to be dependent on conditions (Ref. 8).
For Group B Streptococcus (GBS, S. agalactiae), cell growth rate was reported to be the principal factor regulating capsular polysaccharide production. Furthermore, the production of type III capsular polysaccharide was shown to occur independently of the growth-limiting nutrient. Higher specific yields (up to about 90 mg/gCDW) were obtained when cells were held at a fast (0.8, 1.4 or 1.6 h) mass doubling time (td) rather than at a slow time (td=2.6 or 11 h) (Refs. 8-10). However, continuous culture is prone to strain stability problems and contamination, and is somewhat expensive due to the continuous feed of medium and nutrients. Therefore, there is a need to find alternatives to continuous culture for the high yield production of capsular polysaccharides in order to overcome the problems with continuous culture that are cited above.
One approach to overcome the drawbacks of continuous culture is exemplified in WO 2007/052168. A complex fed batch fermentation process has been developed to maintain a nutritional environment and a growth rate favorable to cps production. This process combines the advantages of batch and continuous techniques, producing high cell densities due to extension of the exponential growth phase and to conditions that control substrate addition during fermentation. However, the complex fed batch technique uses software with a complex algorithm to manage the fermentation. Furthermore, a robust and cost-effective production process in compliance with Good Manufacturing Practices is necessary to generate material to support clinical trials. Therefore, there is an urgent need to simplify the fed batch fermentation process for large-scale production.
In addition to a need for simplified fermentation protocols, there is a need for simplified purification protocols that can be used in the large-scale production of capsular polysaccharides post-fermentation. The approach exemplified in WO 2007/052168 is based on the method disclosed in WO 2006/082527, which includes extraction, alcoholic precipitation, diafiltration, cationic detergent treatment, and re-solubilization. This procedure is highly efficient and typically yields a preparation of capsular polysaccharide that is approximately 80% pure. However, the step of cationic detergent treatment results in precipitation of the capsular polysaccharide. The subsequent separation of the precipitate from the supernatant (e.g. by centrifugation) and re-solubilization is laborious and may result in loss of capsular polysaccharide, thereby reducing yield. The efficiency of the cationic detergent treatment may also be dependent on the initial purity of the capsular polysaccharide. The lower the initial purity of the capsular polysaccharide, the less efficient the cationic detergent treatment may be, further limiting yield. Therefore, there is a need for a simplified purification procedure that will produce higher levels of purity with fewer complicated and/or expensive purification steps. There is also a need for a purification procedure that provides a good yield of capsular polysaccharide whatever the initial purity of the polysaccharide.